A new, simple and precise method using ion-pair high-performance liquid chromatography was developed for the determination of glycyrrhizin in Glycyrrhizae Radix. A reversed-phase system was used, consisting of an ODS column with water and methanol (45 : 55) containing 5mM tetra-n-butylammonium hydroxide and adjusted to pH 6.0 by phosphoric acid as the mobile phase. A peak (peak I) which had the same retention time as glycyrrhizin in the established ion-suppression method was separated by the ion-pair method. The analytical results of glycyrrhizin measured by this method were lower by 6.2% to 10.8% than those obtained by the ion-suppression method.